Abstract

Background

Screening with combined cytologic and HPV testing has led to the highest number of
excessive colposcopic referrals due to high false positive rates of the current HPV
testing in the USA. How best to capitalize on the enhanced sensitivity of HPV DNA
testing while minimizing false-positive results from its lower specificity is an important
task for the clinical pathologists.

Methods

The HPV L1 gene DNA in liquid-based Pap cytology specimens was initially amplified
by the degenerate MY09/MY11 PCR primers and then re-amplified by the nested GP5+/GP6+
primers, or the heminested GP6/MY11, heminested GP5/MY09 primers or their modified
equivalent without sample purification or DNA extraction. The nested PCR products
were used for direct automated DNA sequencing. A 34- to 50-base sequence including
the GP5+ priming site was selected as the signature sequence for routine genotyping
by online BLAST sequence alignment algorithms.

Results

Of 3,222 specimens, 352 were found to contain HPV DNA, with 92% of the positive samples
infected by only 1 of the 35 HPV genotypes detected and 8% by more than 1 HPV genotype.
The most common genotype was HPV-16 (68 isolates), followed by HPV-52 (25 isolates).
More than half (53.7%) of the total number of HPV isolates relied on a nested PCR
for detection although the majority of HPV-16, -18, -31, -33 -35 and -58 isolates
were detected by a single MY09/MY11 PCR. Alignment of a 34-base sequence downstream
of the GP5+ site failed to distinguish some isolates of HPV-16, -31 and -33. Novel
variants of HPV with less than "100% identities" signature sequence match with those
stored in the Genbank database were also detected by signature DNA sequencing in this
rural and suburban population of the United States.

Conclusion

Laboratory staff must be familiar with the limitations of the consensus PCR primers,
the locations of the signature sequence in the L1 gene for some HPV genotypes, and
HPV genotype sequence variants in order to perform accurate HPV genotyping.